(1) Field of the Invention
The present invention relates to a DNA encoding mannose 6-phosphate reductase (M6PR) which is part of the pathway which forms mannitol in plants. The DNA encoding M6PR was isolated from celery. When the DNA is transformed into a plant the resulting transformed plant can be more tolerant to environmental stresses in the form of dehydration, salinity and drought.
(2) Description of Related Art
In many plants, sucrose and starch are the primary products of photosynthetic carbon assimilation. In other species, however, acrylic polyols (e.g., sorbitol, mannitol) can also be primary products and account for between 15 and 60% of the assimilated carbon, depending on the species (Loescher, W. H., Physiol. Plantarum 70:553-557 (1987); Fox, T. C., et al Plant Physiology 82:307-311 (1986); Flora and Madore Planta 189:484-490 (1993); Loescher, W. H. and Everard, J. D., Photoassimilate Distribution in Plants and Crops, 185-207 (1996)), the stage of leaf development, (Davis, J. M., et al., Plant Physiol, 86:129-133 (1988) and environmental factors (e.g., salinity (Everard, J. D., et al., Plant Physiol, 106:281-292 (1994)) and water stress; Escobar-Gutierrez, A., et al., Plant Physiol suppl 105: abstract 575 (1994)). The influence of developmental and environmental factors suggest that the partitioning of photoassimilates between sugar alcohols, sucrose and starch is under strict metabolic control. This is consistent with the complexity and diversity of the control mechanisms known to govern sucrose and starch synthesis in species that do not synthesize sugar alcohols (Quick, W. P. and Schaffer, A. A., Sucrose metabolism in sources and sinks. In: Photoassimilate distribution in plants and crops: source-sink relationships. Zamski, E., and Schaffer A., (eds), Marcel Dekker, Inc.: pp 115-156 (1996); and Preiss, J. and Sivak, M. N., Starch synthesis in sinks and sources. In: Photoassimilate distribution in plants and crops: source-sink relationships. Zamski, E., and Schaffer A., (eds), Marcel Dekker, Inc.: pp 63-96 (1996)). There is, however, almost no equivalent information on the mechanisms by which polyol metabolism is regulated or integrated with these other pathways in sugar alcohol synthesizing species. Such mechanisms are of more than just esoteric interest since: (a) an estimated 30% of global annual carbon assimilation results in polyol production (Bieleski, R. L., Sugar alcohols. In: F. A. Loewus and W. Tanner, eds., Plant Carbohydrates I. Intracellular Carbohydrates, Encyc. Plant Physiol. Vol. 13A, New Series, Springer-Verlag, NY, pp. 158-192 (1982)), (b) many polyol producing species are of considerable economic value, and (c) substantial correlative evidence shows that polyols accumulate in higher plants subjected to stresses mediated at the cellular level by changes in water activity, suggesting a role in stress tolerance (Bohnert, Hans J., et al., The Plant Cell, 7:1099-1111 (1995); Ahmad, I., et al., New Phytol 82:671-679 (1979); Hirai, M., Plant Cell Physiol. 24:925-931 (1983); Everard, J. D., et al., Plant Physiol 106:281-292 (1994); Stoop, J. M. H., et al., Plant Physiol. 106:503-511 (1994)).
In recent years major advances in the understanding of sugar alcohol metabolism in higher plants have been facilitated by the detection, characterization, and purification of several key enzymes (see Loescher and Everard, Photoassimilate Distribution in Plants and Crops, 185-207 (1996) for a review). The successful cloning of genes for NADP-dependent sorbitol 6-phosphate reductase (Kanayama, Y., et al., Plant Physiology 100:1607-1608 (1992)), mannose 1-oxidoreductase (Williamson, J. D., et al., Proc. Natl. Acad. Sci. 92:7148-7152 (1995)) and the introduction of heterologous genes, which confer sugar alcohol synthesis to plants that normally do not produce them (Tarczynski, M. C., et al., Science 259:508-510 (1993); Thomas, J. C., et al., Plant Cell and Environ 18:801-806 (1995); Tao, R., et al., Plant Cell Physiol 36:525-532 (1995)), now provide powerful tools with which to study sugar alcohol biochemistry and physiology. For example, Tarczynski, M. C., et al. (Science 259:508-510 (1993)), and Thomas, J. C., et al. (Plant Cell and Environ 18:801-806 (1995)) have recently shown that transgenic tobacco and Arabidopsis plants expressing the bacterial mannitol dehydrogenase (mtlD) gene not only produce low levels of mannitol, but also exhibit enhanced sodium chloride tolerance. In another study the inhibitory effects of 300 mM NaCl on the growth of celery suspension cultures were substantially reduced when mannitol, rather than sucrose, was included as the sole carbon source (Pharr, D. M., et al., Plant Physiology 180-194 (1995)). Such studies convincingly demonstrate that polyols confer some stress protection but give little insight into the underlying mechanisms. Mechanisms are beginning to be elucidated however, mannitol accumulation in salt stressed celery plants has been associated with a down regulation (at both the mRNA and protein levels) of mannitol dehydrogenase (MTD), a key catabolic enzyme (Williamson, J. D., et al., Proc. Natl. Acad. Sci., 92:7148-7152 (1995)), although, enhanced de novo synthesis is also undoubtedly involved in this acclimation response (Everard, J. D., Plant Physiol. 106:281-292 (1994); Loescher, W. H., et al., Plant Physiology, 170-178 (1995)). Other evidence showing a possible role for polyols in stress metabolism in higher plants includes the identification of an aldose reductase in Craterostigma leaves and barley embryos that accumulates when these tissues undergo desiccation (Bartels, D., et al., EMBO J 10:1037-1043 (1991)).
A pathway for mannitol synthesis in higher plants has been established in celery (Rumpho, M. E., et al., Plant Physiol. 73:869-873 (1983)) and appears to be present in other mannitol synthesizing species (Harloff, H. J., et al., J. Plant Physiol 141:513-520 (1993)). Biosynthesis involves three unique enzymatic steps consisting of an isomerization (F6P to mannose 6-P, mediated by mannose 6-P isomerase), a reduction (mannose 6-P to mannitol 1-P by mannose 6-P reductase (M6PR)) and a dephosphorylation (mannitol 1-P to mannitol, by mannitol 1-P phosphatase). Radiotracer studies and kinetic analyses indicate that M6PR plays a regulatory role in this pathway. This enzyme has been purified and partially characterized (Loescher, W. H., et al., Plant Physiol. 98:1396-1402 (1992)).
U.S. Pat. No. 5,268,288 to Pharr describes mannitol oxidoreductase protein. The enzyme converts mannitol to mannose in plants. It is thus different from the present invention which relates to mannitol production. The patent describes various recombinant techniques useful in the present invention. U.S. Pat. No. 5,492,820 to Sonnewald et al describes plasmids (vectors) for producing recombinant plants with altered sugar expression. The disclosure of such vectors is incorporated into the disclosure of the present application as well.
It is therefore an object of the present invention to provide DNA encoding mannose 6-phosphate reductase (M6PR) which is useful in producing plasmids and transgenic plants with increased tolerance to environmental stresses, particularly salinity. These and other objects will become increasingly apparent by reference to the following description and the drawings.